1. Field of the Invention
The invention relates to the areas of microbial genetics and recombinant DNA technology. More specifically, the present invention relates to the fermentative production of amino acids. The invention provides microorganisms useful for the production of amino acids, methods to increase the production of amino acids and processes for the production of amino acids.
2. Related Art
The production of amino acids through fermentation enables inexpensive production from cheap carbon sources such as molasses, acetic acid and ethanol. Following the recognition that Corynebacteria were useful for the industrial production of amino acids (S. Kinoshita et al., Proceedings of the International Symposium on Enzyme Chemistry 2: 464-468 (1957)), commercial production of amino acids by fermentative processes was made more possible with the isolation of mutant strains. Microorganisms employed in microbial processes for amino acid production may be divided into 4 classes: wild-type strain, auxotrophic mutant, regulatory mutant and auxotrophic regulatory mutant (K. Nakayama et al, in NUTRITIONAL IMPROVEMENT OF FOOD AND FEED PROTEINS, M. Friedman, ed., (1978), pp. 649-661). The stereospecificity of the amino acids produced by fermentation makes the process advantageous compared with synthetic processes; amino acids produced by microbial process are generally the L-form.
L-lysine is one example of an amino acid produced by industrial fermentation. Commercial production of this essential amino acid is principally done utilizing the gram positive Corynebacterium glutamicum, Brevibacteriumflavum and Brevibacterium lactofermentum (Kleemann, A., et. al., Amino Acids, in ULLMAN's ENCYCLOPEDIA of INDUSTRIAL CHEMISTRY, vol. A2, pp. 57-97, Weinham: VCH-Verlagsgesellschaft (1985)); cumulatively, these three organisms presently account for the approximately 250,000 tons of L-lysine produced annually.
Given the economic importance of L-lysine production by fermentive processes, it would be beneficial to increase the total amount produced while simultaneously decreasing production costs. To that end, the biochemical pathway for L-lysine synthesis has been intensively investigated in Corynebacterium (recently reviewed by Sahm et al., Ann. N. Y. Acad. Sci. 782: 25-39 (1996)). Entry into the lysine pathway begins with L-aspartate (see FIG. 1), which itself is produced by transamination of oxaloacetate. A special feature of C. glutamicum is its ability to convert the lysine intermediate piperideine 2,6-dicarboxylate to diaminopimelate by two different routes, i.e. by reactions involving succinylated intermediates or by the single reaction of diaminopimelate dehydrogenase. Overall, carbon flux into the pathway is regulated at two points: first, through feedback inhibition of aspartate kinase by the levels of both L-threonine and L-lysine; and second through the control of the level of dehydrodipicolinate synthase. Therefore, increased production of L-lysine may be obtained in Corynebacteria by deregulating and increasing the activity of these two enzymes.
In addition to the biochemical pathway leading to L-lysine synthesis, recent evidence indicates that consideration of lysine transport out of cells into the media is another condition to be considered in the development of lysine over-producing strains of C. glutamicum. Studies by Krämer and colleagues indicate that passive transport out of the cell, as the result of a leaky membrane, is not the sole explanation for lysine efflux; their data suggest a specific carder with the following properties: (1) the transporter possesses a rather high Km value for lysine (20 mM); (2) the transporter is an OH− symport system (uptake systems are H+ antiport systems); and (3) the transporter is positively charged, and membrane potential stimulates secretion (S. Bröer and R. Krämer, Eur. J. Biochem. 202: 137-143 (1991).
Several fermentation processes utilizing various strains isolated for auxotrophic or resistance properties are known in the art for the production of L-lysine: U.S. Pat. No. 2,979,439 discloses mutants requiring homoserine (or methionine and threonine); U.S. Pat. No. 3,700,557 discloses mutants having a nutritional requirement for threonine, methionine, arginine, histidine, leucine, isoleucine, phenylalanine, cystine, or cysteine; U.S. Pat. No. 3,707,441 discloses a mutant having a resistance to a lysine analog; U.S. Pat. No. 3,687,810 discloses a mutant having both an ability to produce L-lysine and a resistance to bacitracin, penicillin G or polymyxin; U.S. Pat. No. 3,708,395 discloses mutants having a nutritional requirement for homoserine, threonine, threonine and methionine, leucine, isoleucine or mixtures thereof and a resistance to lysine, threonine, isoteucine or analogs thereof; U.S. Pat. No. 3,825,472 discloses a mutant having a resistance to a lysine analog; U.S. Pat. No. 4,169,763 discloses mutant strains of Corynebacterium that produce L-lysine and are resistant to at least one of aspartic analogs and sulfa drugs; U.S. Pat. No. 5,846,790 discloses a mutant strain able to produce L-glutamic acid and L-lysine in the absence of any biotin action-supressing agent; and U.S. Pat. No. 5,650,304 discloses a strain belonging to the genus Corynebacterium or Brevibacterium for the production of L-lysine that is resistant to 4-N-(D-alanyl)-2,4-diamino-2,4-dideoxy-L-arabinose 2,4-dideoxy-L-arabinose or a derivative thereof.
More recent developments in the area of L-lysine fermentive production in Corynebacteria involve the use of molecular biology techniques to augment lysine production. The following examples are provided as being exemplary of the art: U.S. Pat. Nos. 4,560,654 and 5,236,831 disclose an L-lysine producing mutant strain obtained by transforming a host Corynebacterium or Brevibacterium microorganism which is sensitive to S-(2-aminoethyl)-cysteine with a recombinant DNA molecule wherein a DNA fragment conferring both resistance to S-(2-aminoethyl)-cysteine and lysine producing ability is inserted into a vector DNA; U.S. Pat. No. 5,766,925 discloses a mutant strain produced by integrating a gene coding for aspartokinase, originating from coryneform bacteria, with desensitized feedback inhibition by L-lysine and L-threonine, into chromosomal DNA of a Coryneform bacterium harboring leaky type homoserine dehydrogenase or a Coryneform bacterium deficient in homoserine dehydrogenase gene.
In addition to L-lysine, Corynebacterium and related organisms are useful for the production of other amino acids, for example the branched chain amino acids L-leucine, L-isoleucine and L-valine. The biochemical pathways leading to branched chain amino acid biosynthesis are also well studied. Carbon flux into the aspartate pathway may be funneled onto the production of L-lysine or L-threonine, which may be utilized for the production of L-isoleucine (FIG. 1B). L-isoleucine is produced from L-threonine in five reactions; the enzymes catalyzing these reactions include: (1) threonine dehydratase; (2) acetohydoxy acid synthase; (3) isomeroreductase; (4) dihydroxy acid dehydratase; and (5) transaminase B. Threonine dehydratase is the only enzyme in this pathway unique to isoleucine synthesis; the other four enzymes are also utilized in the production of the other branched chain amino acids, valine and leucine. Carbon flux from threonine to isoleucine is controlled by threonine dehydratase and acetohydoxy acid synthase (AHAS). With the cloning of genes encoding the enzymes of the isoleucine pathway (ilvA, ilvB, ilvC, ilvD and ilvE) in Corynebacterium (C. Cordes et al., Gene 112:113-116 (I 992); B. Möckel et al., J. Bacteriology 174: 8065-8072 (1992); and C. Keilhauer et al., J. Bacteriology 175: 5595-5603 (1993)), recombinant DNA techniques may be applied to generate novel strains.
Improvements in the production of the amino acids L-isoleueine, L-leucine and L-valine by increasing the activity of enzymes in the branched chain amino acid biosynthetic pathway have been described. Additionally, improvements in the production of branched chain amino acids by improving the acetohydroxy acid synthase (AHAS) activity encoded by the ilvBN operon have been described. (see generally H. Sahm et al., Ann. N. Y.. Acad. Sci. 782:25-39 (1996)).
Exemplary processes for the production of branched chain amino acids include the following: U.S. Pat. No. 5,188,948 discloses a fermentation process for producing L-valine utilizing a microorganism that is resistant to a polyketide; U.S. Pat. No. 5,521,074 discloses a process for producing L-valine utilizing a microorganism which belongs to the genus Corynebacterium or Brevibacterium, which exhibits a) an ability to produce L-valine, b) resistance to L-valine in a medium containing acetic acid as a sole carbon source, and c) sensitivity to a pyruvic acid analog in a medium containing glucose as a sole carbon source; U.S. Pat. No. 4,601,983 discloses a genetic sequence coding for the production of a protein having the activity of homoserine dehydrogenase capable of replication in coryneform bacteria and used to produce L-threonine and L-isoleucine; U.S. Pat. No. 4,442,208 discloses a fermentation process for the production of L-isoleucine utilizing a Brevibacterium or Corynebacterium strain obtained by recombinant DNA techniques that is resistant to α-amino-β-hydroxy valeric acid; U.S. Pat. No. 4,656,135 discloses a process for producing L-isoleucine, which comprises culturing a microorganism belonging to the genus Brevibacterium or the genus Corynebacterium which has a methyllysine resistance or α-ketomalonic acid resistance and which is capable of producing L-isoleucine in a liquid medium, and obtaining the accumulated L-isoleucine from said medium; U.S. Pat. No. 5,118,619 discloses a method for the fermentative production of L-isoleucine from D,L-α-hydroxybutyrate by means of mutants that utilize D-lactate; U.S. Pat. No. 5,763,231 discloses a process for producing L-leucine, which includes incubating a strain of the genus Corynebacterium, Escherichia, Brevibacterium, or Microbacterium in a culture medium and reacting the resulting cells with saccharides and acetic acid or its salt to form and accumulate L-leucine in the reaction solution; and U.S. Pat. No. 3,970,519 discloses strains that resist feedback inhibition by leucine or its analogs and require at least one of isoleucine, threonine or methionine as a growth nutriment to produce L-leucine.
Improvements in the production of amino acids by decreasing the production of valine have not been described.
Improvements in the production of amino acids by decreasing AHAS activity have not been described.